Conjunctival epithelial cell differentiation on amniotic membrane

Abstract

Purpose: Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation.

Methods: Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy.

Results: In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid.

Conclusions: RCEs cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further.